Nitric oxide (NO.cndot.) is synthesized from the amino acid L-arginine by a family of enzymes termed nitric oxide synthases. Its small size and its unpaired electron (denoted.cndot.), make it a highly reactive and locally diffusible free radical. Since the discovery in 1987 that endothelium derived relaxing factor (EDRF) is, in fact, NO.cndot., (Palmer, R. M. J. et al., 1988, Nature 333:664-6; Palmer, R. M. J. et al., 1987, Nature, 327:524-527) it has become evident that NO.cndot. is a widely distributed and multi-functional intra- and intercellular messenger. There is strong evidence that NO.cndot. synthesized by vascular endothelium is responsible for the regulation of blood pressure and the control of platelet aggregation (Mollace, V. et al., 1990, Biochem. Biophy. Res. Comm., 172:564-9; Rand, M. J. et al., 1992, Clin. Exp. Phamacol. Physiol., 19:147-69; Szabo, C. et al., 1993, Circulation Res., 73:73), and may be involved in vascular injury caused by tissue deposition of immune complexes (Mulligan et al., Proc. Natl. Acad. Sci. USA, 88:6338, 1991). In the central nervous system, nitric oxide is thought to be a neurotransmitter involved in memory and motor function (Bult, H. et al., 1990, Nature, 345:346-347; Dawson, T. M. et al., 1991, Proc. Natl. Acad. Sci. USA, 88:7797-7801; Giovanelli, J. et al., 1991, Proc. Natl. Acad. Sci. USA, 88:7091-7095; Moroz, L. L. et al., 1993, Neuroreport, 4:643-6; Knowles, R. G. et al., 1989, Proc. Natl. Acad. Sci. USA, 86:5159-5162; Snyder, S. H., 1993, Nature, 364:577-632). In the peripheral nervous system, a widespread network of nonadrenergic, noncholinergic nerves use nitric oxide to modulate gastrointestinal, bladder and (corpus cavernosum relaxation and erection. (Moncada, S. et al., 1993, New Engl. J. Med., 329 (27):2002-12; Ward, S. M. et al., 1992, Am. J. Physiol, 263:G277-84).
NO.cndot. is produced in large amounts by an inducible isoform of nitric oxide synthase in macrophages, neutrophils, lymphocytes and peripheral-blood monocytes during immunological reactions and septic shock. (Hevel, J. M. et al., 1991, J. Biol. Chem., 266:22789-22791; Hibbs, J. B. et al, 1988, Biochem. Biophys. Res. Comm., 157:87-94; Nathan, C. F. et al., 1991, Curr. Opin. Immunol., 3:65-70; Salvemini, D. et al., 1990, Biochem. Biophys. Res. Comm., 169:596-601; Szabo, C. et al., 1993, Circulation Res., 73:73). NO.cndot. is also produced in keratinocytes in response to inflammatory mediators (Reck et al., J. Biol. Chem., 267:21277, 1992.) There is also an inducible form of nitric oxide synthase in cartilage. (Murrell, G. A. C. et al., 1994, International, Business Communications 3rd Symposium on Nitric Oxide: Palmer, R. M. et al., 1993, Biochem. Biophys. Res. Comm., 193:398-405; Stadler, J. et al., 1991, J. Immunol, 147:3915-20) Constitutive, Ca.sup.++ dependent isoforms of the enzyme found in the brain and blood vessels release NO.cndot. at low, relatively stable concentrations.
The present invention relates to a previously uncharacterized role for NO in wound healing. Wound healing involves the recruitment of inflammatory cells, followed by fibroblasts, to the site of the wound, where collagen and other connective tissue elements are deposited. The collagen fibers then gradually realign to resemble the original connective tissue (e.g. tendon, ligament, skin.) The ability to regulate this process locally and specifically would be of considerable therapeutic importance e.g. after surgery or trauma. Furthermore, in certain pathological situations, such as arthrofibrosis, Dupuytren's contracture, peritoneal adhesions, frozen shoulder, scleroderma, and keloid formation, over-expression, and sometimes normal expression, of the repair mechanisms has negative consequences, and it would be desirable to selectively suppress this response.
Conversely, there are many situations in which the healing response in wound healing is delayed or inhibited e.g. in patients with systemic diseases such as liver failure, renal impairment, diabetes, peripheral vascular disease, or in patients taking drugs that inhibit healing e.g. corticosteroids or immunosuppressive agents. In these cases, additional exogenous NO.cndot. may enhance the healing response.
Therefore, there is a need in the art for methods and compositions to influence wound healing in a temporally and spatially regulatable manner.